A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice.
The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag. Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv-treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.
Lamarre
A
A
Laboratory of Neuroimmunovirology, Virology Research Center, Institut Armand-Frappier, Université du Québec, Laval, Canada.
Yu
M W
MW
Chagnon
F
F
Talbot
P J
PJ
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000008:administration & dosage / Q000276:immunology*; D001483:Base Sequence; D017934:Coronavirus / Q000276:immunology*; D018352:Coronavirus Infections / Q000276:immunology* / Q000517:prevention & control; D004926:Escherichia coli; D007135:Immunoglobulin Variable Region / Q000008:administration & dosage / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000276:immunology
D000914:Antibodies, Viral; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins